利用大肠杆菌高效合成L-异亮氨酸的系统代谢工程研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-0695

摘要/Abstract

摘要：

目的】 L-异亮氨酸属于必需氨基酸，在医药、食品、农业等领域应用广泛。针对现有L-异亮氨酸生产菌株存在的合成效率偏低、发酵周期长、不稳定等不足，利用系统代谢工程构建L-异亮氨酸高效合成菌株。 方法】 以前期构建的L-异亮氨酸合成菌株ISO-2为研究对象，通过提高草酰乙酸和L-天冬氨酸供应、解除L-异亮氨酸的反馈抑制作用、增强L-异亮氨酸合成代谢流、平衡辅酶水平、构建柠苹酸途径以及强化输出等策略提升L-异亮氨酸合成效率。 结果】 过表达ppc、pycA、aspC和aspA有效提升了草酰乙酸和L-天冬氨酸供应，菌株YL-4的L-异亮氨酸产量达到6.98 g/L。过表达解除反馈抑制的苏氨酸脱水酶编码基因ilvA^YI和ilvD以及NADH依赖的二羟酸还原异构酶和亮氨酸脱氢酶编码基因ilvC^EM和bcd，菌株YL-8的L-异亮氨酸产量提升35.4%。过表达cimA和leuBCD构建柠苹酸途径，菌株YL-12的L-异亮氨酸产量提高至11.03 g/L，其副产物L-缬氨酸积累量降低至0.10 g/L。敲除iclR激活乙醛酸循环并利用自调节启动子P _fliA 动态调控α-脱氢酶编码基因sucAB转录，菌株YL-14的L-异亮氨酸产量提高至11.93 g/L。在此基础上，敲除L-异亮氨酸摄入蛋白编码基因brnQ、过表达其输出载体编码基因ygaZH，菌株YL-16经发酵44 h，L-异亮氨酸产量和转化率分别达到49.73 g/L和0.33 g/g葡萄糖。 结论】 利用系统代谢工程选育出一株合成效率高、发酵周期短且稳定的L-异亮氨酸生产菌株。所用策略可为天冬氨酸族氨基酸及分支链氨基酸菌株的构建提供参考。

关键词: L-异亮氨酸, 大肠杆菌, 代谢工程, 代谢流, 动态调控

Abstract:

Objective L-isoleucine, an essential amino acid, is widely used in pharmaceutical, food, and agricultural industries. To address the limitations of the current industrial strains used for scale-up production of L-isoleucine, such as low synthesis efficiency, prolonged fermentation period, and instability, this study is aimed to construct a highly efficient L-isoleucine-producing strain through systems metabolic engineering. Method ISO-2, an L-isoleucine -producing strain developed previously, was used as the parental strain. Multiple strategies were implemented to enhance its L-isoleucine biosynthesis: Strengthening oxaloacetate and L-aspartate supply, alleviating feedback inhibition of L-isoleucine, enhancing metabolic flux for L-isoleucine synthesis, balancing cofactor levels, introducing citramalate pathway, and reinforcing L-isoleucine efflux. Result The overexpression of ppc, pycA, aspC, and aspA significantly improved oxaloacetate and L-aspartate supply, achieving L-isoleucine titer of 6.98 g/L in strain YL-4. The co-expression of feedback-resistant threonine dehydratase encoding gene ilvA^YI and ilvD, together with NADH-dependent enzymes encoding genes (ilvC^EM and bcd) increased L-isoleucine production by 35.4% in strain YL-8. Implementing the citramalate pathway by cimA and leuBCD elevated L-isoleucine production to 11.03 g/L, and reduced L-valine accumulation to 0.10 g/L in strain YL-12. Knocking out iclR to activate glyoxylate cycle and dynamically regulating sucAB (encoding α-ketoglutarate dehydrogenase) using the auto-regulatory promoter P _fliA boosted L-isoleucine production to 11.93 g/L. Finally, by deleting brnQ (encoding L-isoleucine transporter) and overexpressing ygaZH (encoding L-isoleucine exporter), strain YL-16 produced 49.73 g/L L-isoleucine, with a yield of 0.33 g/g glucose. Conclusion An L-isoleucine hyperproducer with enhanced efficiency and shortened fermentation period was successfully developed via systems metabolic engineering. This combinatorial strategy provides a valuable reference for engineering strains for producing aspartate-family and branched-chain amino acids.

Key words: L-isoleucine, Escherichia coli, metabolic engineering, metabolic flux, dynamic regulation

魏敏华, 李晓童, 姜亚文, 周飘飘, 汪凯, 孙浩, 芦楠, 张成林. 利用大肠杆菌高效合成L-异亮氨酸的系统代谢工程研究[J]. 生物技术通报, 2025, 41(11): 110-120.

WEI Min-hua, LI Xiao-tong, JIANG Ya-wen, ZHOU Piao-piao, WANG Kai, SUN Hao, LU Nan, ZHANG Cheng-lin. Systems Metabolic Engineering for Highly Efficient L-isoleucine Production in Escherichia coli[J]. Biotechnology Bulletin, 2025, 41(11): 110-120.

图/表 14

表1 L-异亮氨酸代谢工程研究现状

Table 1 Recent advances in metabolic engineering for L-isoleucine production

底盘细胞 Chassis	主要策略 Main strategies	产量 Titer （g/L）	转化率 Yield （g/g Glucose）	周期 Period（h）	发酵强度Productivity（g/L/h）	参考文献 Reference
谷氨酸棒杆菌 Corynebacterium glutamicum	敲除alaT、brnQ和alr；过表达ilvBN^M、ppnK、lrp、brnFE和ilvA^M	32.1	0.18	72	0.45	［11］
大肠杆菌 Escherichia coli	过表达ilvA^fbr、ilvIH、thrABC、ygaZH、ilvEDA和ilvC	9.5	0.14	60	0.16	［4］
	过表达ilvIH1、ygaZH和CgilvA1；敲除poxB、pflB、ldhA、adhE和tdcC	49.3	0.32	48	1.03	［13］
	敲除brnQ、livj、livk和ackA；过表达ygaZH、cimA3.7、GsleuCD、AfleuB ilvIH^fbr、ilvC^NADH、ilvD、LsleuDH和dcuD；	56.6	0.21	34	1.66	［14］
	过表达ilvA^YI、ilvBN^YI、thrA^fbrBC、pntAB、aspA、brnFE、bcd、metA^G189C和metB^M；敲除brnQ	51.5	0.29	44	1.13	［15］
	过表达thrA^fbrBC、ilvGM（AT）、asd、aspA、ilvEDA^fbr、rhtC和pntAB；敲除pykF和ptsG	5.42	0.40	12	0.83	［16］

图1 L-异亮氨酸合成途径及本研究主要策略

Fig. 1 Pathway for synthesis of L-isoleucine and main strategies used in this study

表2 菌株和质粒

Table 2 Strains and plasmids

菌株/质粒 Strains/Plasmids	特性 Characteristics	来源 Sources
菌株
E. coli DH5α	F-， Δ（lacZYA-argF）U169 recA1endA1 hsdR17	实验室保存
ISO-2	E. coli W3110 ΔlacI P _thrABC -thrA：：P _trc -thrA^fbrylbE：：P _trc -thrA^fbrBC-T _trcyjiP：：P _trc -ilvA^YI-T _trcyncI：：P _trc -ilvBN^YI-T _trc	［15］
YL-1	ISO-2 tehB：：P _trc -ppc	本研究
YL-2	YL-1 yciQ：：P _trc -pycA	本研究
YL-3	YL-2 yeeP：：P _trc -aspC	本研究
YL-4	YL-3 ygaY：：P _trc -aspA	本研究
YL-5	YL-4 yjgX：：P _trc -ilvA^YI	本研究
YL-6	YL-5 yghX：：P _trc -ilvD	本研究
YL-7	YL-6 P _ilvC -ilvC：：P _trc -ilvC^EM	本研究
YL-8	YL-7 P _ilvE -ilvE：：P _trc -bcd	本研究
YL-9	YL-8 yeeL：：P _tr -leuBCD	本研究
YL-10	YL-9 gapC：：P_{BBa_J23110}-cimA	本研究
YL-11	YL-9 gapC：：P_{BBa_J23104}-cimA	本研究
YL-12	YL-9 gapC：：P_{BBa_J23119}-cimA	本研究
YL-13	YL-12 △iclR	本研究
YL-14	YL-13 P _sucAB ：：P _fliA	本研究
YL-15	YL-14 △brnQ	本研究
YL-16	YL-15 yjiV：：P _trc -ygaZH	本研究
质粒
pREDCas9	Spe^R，Cas9和λ Red表达质粒	［17］
pGRB	Amp^R，gRNA表达质粒	［17］
pGRB-tehB	pGRB含tehB靶点sgRNA	本研究
pGRB-yciQ	pGRB含yciQ靶点sgRNA	本研究
pGRB-yeeP	pGRB含yeeP靶点sgRNA	本研究
pGRB-ygaY	pGRB含ygaY靶点sgRNA	本研究
pGRB-yjgX	pGRB含yjgX靶点sgRNA	本研究
pGRB-yghX	pGRB含yghX靶点sgRNA	本研究
pGRB-ilvC	pGRB含ilvC靶点sgRNA	本研究
pGRB-ilvE	pGRB含ilvE靶点sgRNA	本研究
pGRB-yeeL	pGRB含yeeL靶点sgRNA	本研究
pGRB-gapC	pGRB含gapC靶点sgRNA	本研究
pGRB-iclR	pGRB含iclR靶点sgRNA	本研究
pGRB-P _sucAB	pGRB含P _sucAB 靶点sgRNA	本研究
pGRB-brnQ	pGRB含brnQ靶点sgRNA	本研究
pGRB-yjiV	pGRB含yjiV靶点sgRNA	本研究

图2 菌株YL-1、YL-2、YL-3和YL-4鉴定图谱A-D分别为菌株YL-1、YL-2、YL-3和YL-4鉴定图谱。M： marker （10 000、 8 000、 7 000、 6 000、 5 000、 4 000、 3 500、 3 000、 2 500、 2 000、 1 500、 1 000、 750、 500、 250 bp，下同）；1：上游同源臂；2：下游同源臂；3：目的基因；4：重叠PCR产物；5：分别以YL-1、YL-2、YL-3或YL-4基因组DNA为模板的PCR扩增产物；6：以出发菌株基因组DNA为模板的PCR扩增产物

Fig. 2 Identification maps of YL-1, YL-2, YL-3 and YL-4A-D: Identification maps of YL-1, YL-2, YL-3 and YL-4. M: DNA marker (10 000, 8 000, 7 000, 6 000, 5 000, 40 00, 3 500, 3 000, 25 00, 2 000, 1 500, 1 000, 750, 500, 250 bp); 1: upstream homology arm; 2: downstream homology arm; 3: target gene; 4: overlapped PCR product; 5: PCR product amplified from genomic DNA of YL-1, YL-2, YL-3 and YL-4, respectively; 6: PCR product amplified from genomic DNA of the starting strain

图3 重编程磷酸烯醇式丙酮酸-丙酮酸-草酰乙酸-天冬氨酸节点对菌株性能的影响

Fig. 3 Effects of reprogramming the phosphoenolpyruvate-pyruvate-oxaloacetate-aspartate node on strains' performances

图4 菌株YL-5-YL-8鉴定图谱A-D分别为菌株YL-5、YL-6、YL-7和YL-8鉴定图谱。M： marker；1：上游同源臂；2：下游同源臂；3：目的基因；4：重叠PCR产物；5：以YL-5、YL-6、YL-7和YL-8基因组DNA为模板的PCR扩增产物；6：以出发菌株基因组DNA为模板的PCR扩增产物

Fig. 4 Identification maps of YL-5-YL-8A-D: Identification maps of YL-5, YL-6, YL-7 and YL-8. M: DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: target gene; 4: overlap PCR product; 5: PCR product amplified from genomic DNA of YL-5, YL-6, YL-7 and YL-8, respectively; 6: PCR product amplified from genomic DNA of the starting strain

图5 强化L-异亮氨酸合成代谢流对菌株性能的影响

Fig. 5 Effects of enhancing L-isoleucine metabolic flux on strains' performances

图6 菌株YL-9-YL-12鉴定图谱A-D分别为菌株YL-9、YL-10、YL-11和YL-12鉴定图谱。M： marker；1：上游同源臂；2：下游同源臂；3：目的基因；4：重叠PCR产物；5：以YL-9、YL-10、YL-11和YL-12基因组DNA为模板的PCR扩增产物；6：以出发菌株基因组DNA为模板的PCR扩增产物

Fig. 6 Identification maps of YL-9-YL-12A-D: Identification maps of YL-9, YL-10, YL-11 and YL-12; M: DNA marker; 1: upstream homology arm; 2: downstream homology arm; 3: target gene; 4: overlap PCR product; 5: PCR product amplified from genomic DNA of YL-9, YL-10, YL-11 and YL-12, respectively; 6: PCR product amplified from genomic DNA of the starting strain

图7 引入柠苹酸途径对菌株性能的影响A：菌株YL-9、YL-10、YL-11和YL-12 L-异亮氨酸产量和生物量；B：菌株YL-8、YL-9、YL-10、YL-11和YL-12胞内α-酮丁酸浓度

Fig. 7 Effects of introducing the citramalate pathway on strains' performancesA: L-Isoleucine production and biomass of strain YL-9, YL-10, YL-11, and YL-12. B: Intracellular α-ketobutyrate concentration in strain YL-8, YL-9, YL-10, YL-11, and YL-12

图8 菌株YL-13和YL-14鉴定图谱A：菌株YL-13鉴定图谱；M：marker；1：上游同源臂U iclR；2：下游同源臂D iclR；3：U iclR -D iclR；4：以YL-13基因组DNA为模板的PCR扩增产物；5：以YL-12基因组DNA为模板的PCR扩增产物。B：菌株YL-14鉴定图谱；M：marker；1：上游同源臂UPsucAB；2：下游同源臂DPsucAB；3：P fliA；4：UPsucAB -P fliA -DPsucAB；5：以YL-14基因组DNA为模板的PCR扩增产物；6： YL-13基因组DNA为模板的PCR扩增产物

Fig. 8 Identification maps of YL-13 and YL-14A: Identification map of YL-13; M: DNA marker; 1: upstream homology arm U iclR; 2: downstream homology arm D iclR; 3: U iclR -D iclR; 4: PCR product amplified from YL-13 genomic DNA; 5: PCR product from YL-12 genomic DNA. B: Identification map of YL-14; M: DNA marker; 1: upstream homology arm UPsucAB; 2: downstream homology arm DPsucAB; 3: P fliA; 4: UPsucAB -P fliA -DPsucAB overlap PCR product; 5: PCR product amplified from YL-14 genomic DNA; 6: PCR product from YL-13 genomic DNA

图9 激活乙醛酸循环并动态弱化TCA循环对菌株性能的影响A：菌株YL-14中α-酮戊二酸脱氢酶相对活性；B：YL-13和YL-14发酵性能

Fig. 9 Effects of activating glyoxylate cycle and dynamically attenuating TCA cycle on strains' performancesA: α-Ketoglutarate dehydrogenase activity in strain YL-14. B: Production performances of YL-13 and YL-14

图10 菌株YL-15和YL-16鉴定图谱A为菌株YL-15鉴定图谱；M： marker；1：上游同源臂U brnQ；2：下游同源臂D brnQ；3： U brnQ -D brnQ；4：以YL-15基因组DNA为模板的PCR扩增产物；5：以YL-14基因组DNA为模板的PCR扩增产物。B为菌株YL-16鉴定图谱；M： marker；1：上游同源臂U yjiV；2：下游同源臂D yjiV；3：P trc -ygaZH-T trc；4：U yjiV -P trc -ygaZH-T trc -D yjiV；5：以YL-16基因组DNA为模板的PCR扩增产物；6：以YL-15基因组DNA为模板的PCR扩增产物

Fig. 10 Identification maps of YL-15 and YL-16A: Identification map of YL-15; M: DNA marker; 1: upstream homology arm U brnQ; 2: downstream homology arm D brnQ; 3: U brnQ -D brnQ; 4: PCR product amplified from YL-15 genomic DNA; 5: PCR product from YL-14 genomic DNA. B: Identification map of YL-16; M: DNA marker; 1: upstream homology arm U yjiV; 2: downstream homology arm D yjiV; 3: P trc -ygaZH-T trc; 4: U yjiV -P trc -ygaZH-T trc -D yjiV; 5: PCR product amplified from YL-16 genomic DNA; 6: PCR product amplified from YL-15 genomic DNA

图11 修饰L-异亮氨酸运输系统对菌株性能的影响A：菌株YL-15和YL-16发酵性能。B：菌株YL-14、YL-15和YL-16胞内L-异亮氨酸浓度

Fig. 11 Effects of modifying L-isoleucine transportation system on production performancesA: Fermatention performances of YL-15 and YL-16. B: Intracellular L-isoleucine levels in YL-14, YL-15, and YL-16

图12 5 L发酵罐中YL-16发酵过程曲线

Fig. 12 Fed-batch fermentation process curve of YL-16 in a 5 L bioreactor

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